CN108152275A - A kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system - Google Patents
A kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system Download PDFInfo
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- CN108152275A CN108152275A CN201711450601.2A CN201711450601A CN108152275A CN 108152275 A CN108152275 A CN 108152275A CN 201711450601 A CN201711450601 A CN 201711450601A CN 108152275 A CN108152275 A CN 108152275A
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Abstract
The invention discloses a kind of hyaluronic acid enzyme assay methods based on electrochemiluminescsystem system, and three (2,2 bipyridyl) chlorination ruthenium systems are hydrated by hyaluronic acid six, construct one for detecting the electrochemiluminescsystem system of hyaluronidase.Hyaluronidase can carry out endonuclease reaction to hyaluronic acid, and the hyaluronic acid fragments after digestion can be entered by centrifugal ultrafiltration in ultrafiltrate together with six three (2, the 2 bipyridyl) ruthenic chlorides of hydration of Electrostatic Absorption on hyaluronic acid fragments.Therefore in the presence of hyaluronidase, the amount that the luminescent substance six in ultrafiltrate is hydrated three (2,2 bipyridyl) ruthenic chlorides can increase, and the electrochemical luminescence intensity of luminescence system can enhance, and the detection to hyaluronidase concentration can be realized based on this.The method of the present invention raw material is easy to get, is easy to operate, taking shorter and high sensitivity, is expected to be used widely in the fields such as life science and clinical medicine detection.
Description
Technical field
The invention belongs to analytical chemistry fields, and in particular to a kind of hyaluronidase detection based on electrochemiluminescsystem system
Method.
Background technology
Hyaluronic acid（HA）It is a kind of linear anionic glycosaminoglycan, the structure of hyaluronic acid is by D-Glucose aldehydic acid and N-
The repetition disaccharide unit composition that acetyl-D-glucose amine is formed.The synthesis and degradation of hyaluronic acid and various bioprocess, such as
Embryo occurs, inflammation, wound healing, and cell Proliferation breaks up and migrates hair that is closely related, and may participating in certain malignant tumours
Exhibition.Hyaluronidase（HAase）It is a kind of endoglucanase, hyaluronic acid can be cut into small fragment, it can be saturating by degrading
Bright matter acid adjusts the transfer of tumour cell.It is reported that the overexpression of hyaluronidase and many malignant tumours such as prostate cancer,
Carcinoma of urinary bladder, the cancer of the brain are related with colorectal cancer.Therefore, hyaluronidase carries out it early stage as potential tumor markers
Detection is of great significance to the clinical diagnosis and treatment of cancer.At this stage, the method that predominantly detects that researcher develops has
Viscosimetry, zymography, turbidimetry, fluorescence method, colorimetric method, immunoassay etc..But these methods be required to it is complicated and very long
Preliminary preparation or valuable cumbersome instrumentation.Therefore, develop a kind of simple and fast hyaluronic acid enzyme assay method
Be there is an urgent need to.
Invention content
The purpose of the present invention is to provide a kind of hyalomitomes easy to operate, high sensitivity based on electrochemiluminescsystem system
The detection method of sour enzyme.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system, includes the following steps：
Step S1：Prepare the electrochemiluminescsystem system that hyaluronic acid-six is hydrated three (2,2- bipyridyls) ruthenic chloride mixed liquors composition；
Step S2：The hyaluronidase of various concentration is added separately in above-mentioned luminescence system, endonuclease reaction is carried out, after reaction
Each mixed solution of gained carries out centrifugal ultrafiltration respectively, collects each ultrafiltrate, and each ultrafiltrate is delayed respectively with positive tripropyl amine (TPA), PBS
Fliud flushing is uniformly mixed, and obtains different mixed systems, is surveyed using by CHI660D electrochemical workstations and the faint chemiluminescences of BPCL
The electrochemical luminescence detecting system for measuring instrument composition measures the electrochemical luminescence signals of each mixed system；
Step S3：According to the electrochemical luminescence signals of each mixed system of collection, standard curve is drawn；
Step S4：Sample to be tested is added in the luminescence system prepared according to step S1, the mixed solution of gained carries out after reaction
Centrifugal ultrafiltration collects ultrafiltrate to be measured, and ultrafiltrate to be measured with positive tripropyl amine (TPA), PBS buffer solution is uniformly mixed, obtains body to be measured
System, uses the electrochemical luminescence detecting system being made of CHI660D electrochemical workstations and the faint chemiluminescence measuring instruments of BPCL
The electrochemical luminescence signals of the system to be measured are measured, which obtains in sample to be tested
Hyaluronidase concentration.
The step S1 is specific as follows：
Step S1-1：The hyaluronic acid solution of 1.4 mg/mL and six three (2,2- bipyridyls) ruthenic chlorides of hydration of 4 mg/mL is molten
Liquid is uniformly mixed according to volume ratio 495: 5, is reacted at room temperature 40-50 minutes, obtains the mixed liquor that volume is V；
Step S1-2：Above-mentioned mixed liquor is placed in super filter tube, is centrifuged 25-35 minutes under 13000-15000rpm rotating speeds, is received
Collect concentrate；
Step S1-3：Above-mentioned concentrate is subjected to centrifuge washing with the PBS buffer solution of a concentration of 10 mM, is repeated 2-3 times, it will most
It is 0.6V that the concentrate obtained eventually is diluted to volume with a concentration of 10 mM, pH PBS buffer solution for being 7.4, obtain hyaluronic acid-
The electrochemiluminescsystem system of six hydration three (2,2- bipyridyls) ruthenic chloride mixed liquors compositions.
The step S2 is specific as follows：
Step S2-1：The hyaluronidase of various concentration is added separately to hyaluronic acid-six and is hydrated three (2,2- bipyridyls) chlorine
In the electrochemiluminescsystem system for changing ruthenium mixed liquor composition, endonuclease reaction is carried out at 37 DEG C 110-130 minutes, it is molten to obtain first
Liquid；
Step S2-2：First solution is placed in super filter tube, is centrifuged, collects ultrafiltrate；
Step S2-3：Ultrafiltrate is uniformly mixed with Tri-n-Propylamine solution, PBS buffer solution, immediately with by CHI660D electrochemistry works
The electrochemical luminescence detecting system for making station and the faint chemiluminescence measuring instrument compositions of BPCL measures its electrochemical luminescence signals.
Further, in step S2, hyaluronidase, hyaluronic acid-six be hydrated three (2,2- bipyridyl) ruthenic chloride mixed liquors,
Tri-n-Propylamine solution, PBS buffer solution amount ratio be L: 8 μ of L: 300 μ of 100 μ L: 2 mL, a concentration of the 10 of the PBS buffer solution
MM, pH 7.4.
The step S4 is specific as follows：
Step S4-1：Sample to be tested is added to hyaluronic acid-six and is hydrated three (2,2- bipyridyls) ruthenic chloride mixed liquors composition
In electrochemiluminescsystem system, reacted 110-130 minutes at 37 DEG C, obtain the first solution；
Step S4-2：First solution is placed in super filter tube, is centrifuged, collects ultrafiltrate；
Step S4-3：Ultrafiltrate is uniformly mixed with Tri-n-Propylamine solution, PBS buffer solution to obtain system to be measured, immediately with by
It is to be measured that the electrochemical luminescence detecting system of CHI660D electrochemical workstations and the faint chemiluminescence measuring instrument compositions of BPCL measures this
The electrochemical luminescence signals of system by the electrochemical luminescence signals combined standard curve, obtain hyaluronidase in sample to be tested
Concentration.
In step step S4, the sample to be tested, hyaluronic acid-six be hydrated three (2,2- bipyridyl) ruthenic chloride mixed liquors,
Tri-n-Propylamine solution, PBS buffer solution amount ratio be L: 8 μ of L: 300 μ of 100 μ L: 2 mL, a concentration of the 10 of the PBS buffer solution
MM, pH 7.4.
The present invention is hydrated three (2,2- bipyridyl) chlorination ruthenium systems, structure using above technical scheme by hyaluronic acid-six
One has been built for detecting the electrochemiluminescsystem system of hyaluronidase.It is anti-that hyaluronidase can carry out digestion to hyaluronic acid
Should, six three (2, the 2- bipyridyl) chlorinations of hydration of hyaluronic acid fragments together with Electrostatic Absorption on hyaluronic acid fragments after digestion
Ruthenium can be entered by centrifugal ultrafiltration in ultrafiltrate.Therefore in the presence of hyaluronidase, the luminescent substance six in ultrafiltrate
The amount of three (2,2- bipyridyl) ruthenic chlorides of hydration can increase, and the electrochemical luminescence intensity of luminescence system can enhance, can be with based on this
Realize the detection to hyaluronidase concentration.
The remarkable advantage of the present invention is：
1st, required raw material are simple and easy to get, do not need to complicated synthesis step.
2nd, it is easy to operate, it is simple to the detection of hyaluronidase and quick without expensive instrument and complicated operation.
3rd, the method for the present invention can be directly used for detection hyaluronidase, from 2 U/mL to the concentration range of 40 U/mL in
Preferable linear response is presented to hyaluronidase.
Description of the drawings
Fig. 1 is the detects schematic diagram of the hyaluronidase of the present invention；
Fig. 2 is the electrochemical luminescence spectrum of various concentration hyaluronidase, adds in the hyaluronidase of various concentration, corresponding to send out
Luminous intensity can change, and the hyaluronidase concentration from a to f is respectively 0 U/mL, 2 U/mL, 10 U/mL, 20 U/mL, 30 U/
mL、40 U/mL；
Fig. 3 is the variation of the corresponding electrochemical luminescence intensity of various concentration hyaluronidase；
Fig. 4 is the detection specific outcome of the method for the present invention.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Solution is prepared：
Hyaluronic acid solution：4.2 mg hyaluronic acids are weighed, 3 mL water dissolutions is added to obtain a concentration of 1.4 mg/ in centrifuge tube
The hyaluronic acid solution of mL is vibrated to hyaluronic acid and is uniformly mixed in the solution, when use be divided into 6 parts it is spare.
Six three (2,2- bipyridyls) chlorination ruthenium solutions of hydration：It weighs 4 mg six and is hydrated three (2,2- bipyridyl) ruthenic chlorides, add 1
ML water dissolutions obtain six three (2,2- bipyridyl) ruthenic chloride solution for standby of hydration of a concentration of 4 mg/mL in centrifuge tube.
1x PBS buffer solution：The 20x PBS buffer solution (200mM) of 2 mL is measured, with the ultrapure water dissolutions of 38 mL, obtains 40
The 1x PBS buffer solution of mL（10mM）It is spare.
Embodiment 1
Hyaluronic acid-six is hydrated the preparation of three (2,2- bipyridyls) ruthenic chloride electrochemiluminescsystem systems
（1）The six of the hyaluronic acid 495 μ L and 4 mg/mL of 1.4 mg/mL is taken to be hydrated three (2,2- bipyridyls) chlorination ruthenium solutions 5
μ L are mixed in centrifuge tube, are reacted at room temperature 45 minutes, are obtained mixed solution；
（2）Obtained mixed solution is placed in super filter tube（Amicon® Ultra-0.5 Centrifugal Filter
Devices）In, it is centrifuged（14000rpm, 30 minutes）, ultrafiltrate is discarded, is obtained containing the hydration of hyaluronic acid-six three
The concentrate of (2,2- bipyridyls) ruthenic chloride compound；
（3）Acquired concentrate is utilized into 1xPBS buffer solutions（10 mM）Carry out centrifuge washing（14000 rpm, 30 minutes）, obtain
The concentrate arrived continues repeated centrifugation and washs 3 times, and the concentrate finally obtained is used 1x PBS buffer solution（pH =7.4）Dilution
To 300 microlitres, the electrochemiluminescsystem system that hyaluronic acid-six is hydrated three (2,2- bipyridyl) ruthenic chloride mixed liquors composition is obtained.
Embodiment 2
The drafting of standard curve
（1）By the hyaluronidase of various concentration（100 µL）Respectively three (2,2- bipyridyls) chlorinations are hydrated with hyaluronic acid-six
The electrochemiluminescsystem system of ruthenium mixed liquor composition is uniformly mixed, and mixed liquor carries out endonuclease reaction 120 minutes at 37 DEG C；
（2）Then each miscible fluid is placed in progress ultrafiltration centrifugation in super filter tube（14000rpm, 30 minutes）, concentrate is discarded,
Obtained ultrafiltrate and the positive tripropyl amine (TPA) solution of 8 μ L and 2 mL 1x PBS buffer solution（pH =7.4）It is placed in luminous pond and mixes
Uniformly；
（3）The electrochemical luminescence being made of CHI660D electrochemical workstations and the faint chemiluminescence measuring instruments of BPCL is used immediately
Detecting system measures the electrochemical luminescence signals in the pond that shines, and detection process uses three-electrode system, is glass-carbon electrode respectively（Work
Make electrode）, silver/silver chloride electrode（Reference electrode）, platinum electrode（To electrode）.
According to the electrochemical luminescence strength signal of collection, monitoring result is recorded.Dependent linearity is fitted according to the reading of record
Equation, obtained linear equation are used for the detection of hyaluronidase concentration in sample to be tested.
As shown in Fig. 2, the concentration with hyaluronidase increases, corresponding electrochemical luminescence intensity also increases therewith.
Hyaluronidase solution is a to f, and the hyaluronidase concentration from a to f is respectively 0 U/mL, 2 U/mL, 10 U/mL, 20 U/
ML, 30 U/mL, 40 U/mL, the luminous intensity of each system can be gradually increased with the increase of hyaluronidase concentration.Therefore,
The present invention can realize the quantitative detection to hyaluronidase concentration.Fig. 3 is the corresponding electrification of various concentration hyaluronidase
Learn the variation of luminous intensity.
Embodiment 3
The measure of hyaluronidase concentration in sample to be tested
（1）By sample to be tested（100 µL）The electricity of three (2,2- bipyridyls) ruthenic chloride mixed liquors composition is hydrated with hyaluronic acid-six
Chemical luminous system is uniformly mixed, and mixed liquor reacts 120 minutes at 37 DEG C；
（2）Then mixed liquor is placed in progress ultrafiltration centrifugation in super filter tube（14000rpm, 30 minutes）, concentrate is discarded, institute
Obtained ultrafiltrate and the positive tripropyl amine (TPA) solution of 8 μ L and 2 mL 1x PBS buffer solution（pH =7.4）It is placed in luminous pond and mixes
It is even；
（3）The electrochemical luminescence being made of CHI660D electrochemical workstations and the faint chemiluminescence measuring instruments of BPCL is used immediately
Detecting system measures the electrochemical luminescence signals in the pond that shines, and detection process uses three-electrode system, is glass-carbon electrode respectively（Work
Make electrode）, silver/silver chloride electrode（Reference electrode）, platinum electrode（To electrode）.
The electrochemical luminescence strength signal of sample to be tested is recorded, substitutes into standard curve, calculates saturating in sample to be tested
The concentration of bright matter acid enzyme.
Embodiment 4
Specific detection
In order to detect the specificity that the method for the present invention detects hyaluronidase, the hyaluronidase used in the present invention is changed
Into other interfering substances, respectively sodium chloride, potassium chloride, glucose, bovine serum albumin, alkaline phosphatase and blank solution, thoroughly
A concentration of 20 U/Ml (0.05 mg/mL) of bright matter acid enzyme, the concentration of other interfering ions is 1 mg/mL.
As shown in figure 3, three (2,2- bipyridyl) ruthenic chloride electrochemiluminescsystem systems are hydrated for hyaluronic acid-six, saturating
Bright matter acid enzyme there are when detect the fluorescence signal that significantly enhances, but the electrochemical luminescence intensity of other interfering substances
It is almost identical with blank solution, this shows that the system responds hyalomitome that is smaller, and being proposed to other interfering substances
Acid-six, which is hydrated three (2,2- bipyridyls) ruthenic chloride electrochemiluminescsystem systems, significant specificity.
Claims (9)
1. a kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system, it is characterised in that：It includes the following steps：
Step S1：Prepare the electrochemiluminescsystem system that hyaluronic acid-six is hydrated three (2,2- bipyridyls) ruthenic chloride mixed liquors composition；
Step S2：The hyaluronidase of various concentration is added separately in above-mentioned luminescence system, endonuclease reaction is carried out, after reaction
Each mixed solution of gained carries out centrifugal ultrafiltration respectively, collects each ultrafiltrate, and each ultrafiltrate is delayed respectively with positive tripropyl amine (TPA), PBS
Fliud flushing is uniformly mixed, and obtains different mixed systems, and the electricity of each mixed system is measured using the faint chemiluminescence measuring instruments of BPCL
Chemiluminescence signal；
Step S3：According to the electrochemical luminescence signals of each mixed system of collection, standard curve is drawn；
Step S4：Sample to be tested is added in the luminescence system prepared according to step S1, the mixed solution of gained carries out after reaction
Centrifugal ultrafiltration collects ultrafiltrate to be measured, and ultrafiltrate to be measured with positive tripropyl amine (TPA), PBS buffer solution is uniformly mixed, obtains body to be measured
System, the electrochemical luminescence signals of the system to be measured are measured using the faint chemiluminescence measuring instruments of BPCL, which is believed
Number combined standard curve, obtains hyaluronidase concentration in sample to be tested.
2. a kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system according to claim 1, feature exist
In：The step S1 is specific as follows：
Step S1-1：The hyaluronic acid solution of 1.4 mg/mL and six three (2,2- bipyridyls) ruthenic chlorides of hydration of 4 mg/mL is molten
Liquid is uniformly mixed according to volume ratio 495: 5, is reacted at room temperature 40-50 minutes, obtains the mixed liquor that volume is V；
Step S1-2：Above-mentioned mixed liquor is placed in super filter tube, is centrifuged, collects concentrate；
Step S1-3：Above-mentioned concentrate is subjected to centrifuge washing with the PBS buffer solution of a concentration of 10 mM, is repeated 2-3 times, it will most
It is 0.6V that the concentrate obtained eventually is diluted to volume with a concentration of 10 mM, pH PBS buffer solution for being 7.4, obtain hyaluronic acid-
The electrochemiluminescsystem system of six hydration three (2,2- bipyridyls) ruthenic chloride mixed liquors compositions.
3. a kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system according to claim 2, feature exist
In：The centrifugal rotational speed of step S1-2 is 13000-15000rpm, and centrifugation time is 25-35 minutes.
4. a kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system according to claim 1, feature exist
In：The step S2 is specific as follows：
Step S2-1：The hyaluronidase of various concentration is added separately to hyaluronic acid-six and is hydrated three (2,2- bipyridyls) chlorine
In the electrochemiluminescsystem system for changing ruthenium mixed liquor composition, endonuclease reaction is carried out at 37 DEG C 110-130 minutes, it is molten to obtain first
Liquid；
Step S2-2：First solution is placed in super filter tube, is centrifuged, collects ultrafiltrate；
Step S2-3：Ultrafiltrate is uniformly mixed with Tri-n-Propylamine solution, PBS buffer solution, immediately with the faint chemiluminescences of BPCL
Measuring instrument measures its electrochemical luminescence signals.
5. a kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system according to claim 4, feature exist
In：The hyaluronidase, hyaluronic acid-six are hydrated three (2,2- bipyridyls) ruthenic chloride mixed liquors, Tri-n-Propylamine solution, PBS
The amount ratio of buffer solution is L: 2 mL of L: 8 μ of L: 300 μ of 100 μ.
6. a kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system according to claim 4, feature exist
In：A concentration of 10 mM, pH 7.4 of the PBS buffer solution.
7. a kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system according to claim 1, feature exist
In：The step S4 is specific as follows：
Step S4-1：Sample to be tested is added to hyaluronic acid-six and is hydrated three (2,2- bipyridyls) ruthenic chloride mixed liquors composition
In electrochemiluminescsystem system, reacted 110-130 minutes at 37 DEG C, obtain the first solution；
Step S4-2：First solution is placed in super filter tube, is centrifuged, collects ultrafiltrate；
Step S4-3：Ultrafiltrate is uniformly mixed with Tri-n-Propylamine solution, PBS buffer solution to obtain system to be measured, uses BPCL immediately
Faint chemiluminescence measuring instrument measures the electrochemical luminescence signals of the system to be measured, by electrochemical luminescence signals combined standard song
Line obtains hyaluronidase concentration in sample to be tested.
8. a kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system according to claim 7, feature exist
In：The sample to be tested, hyaluronic acid-six are hydrated three (2,2- bipyridyls) ruthenic chloride mixed liquors, Tri-n-Propylamine solution, PBS and delay
The amount ratio of fliud flushing is L: 2 mL of L: 8 μ of L: 300 μ of 100 μ.
9. a kind of hyaluronic acid enzyme assay method based on electrochemiluminescsystem system according to claim 7, feature exist
In：A concentration of 10 mM, pH 7.4 of the PBS buffer solution.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111443007A (en) * | 2020-04-13 | 2020-07-24 | 厦门大学附属厦门眼科中心有限公司 | Detection method for measuring concentration of hyaluronidase based on flow velocity of hydrogel composite membrane |
| CN112964768A (en) * | 2021-03-23 | 2021-06-15 | 海南微氪生物科技股份有限公司 | Bst DNA polymerase electrochemiluminescence determination method |
| CN112980925A (en) * | 2021-04-06 | 2021-06-18 | 厦门大学附属厦门眼科中心有限公司 | MicroRNA detection method based on DNA hydrogel electrochemiluminescence system |
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| CN102680456A (en) * | 2011-03-16 | 2012-09-19 | 北京联众泰克科技有限公司 | ECLI (Electro ChemiLuminescence Immunoassay) determining method |
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| CN101057145A (en) * | 2004-06-23 | 2007-10-17 | 德克萨斯***大学 | Methods and compositions for the detection of biological molecules using a two particle complex |
| CN102262051A (en) * | 2010-05-25 | 2011-11-30 | 香港城市大学 | Optical sensing devices and methods for detecting samples using the same |
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| CN111443007A (en) * | 2020-04-13 | 2020-07-24 | 厦门大学附属厦门眼科中心有限公司 | Detection method for measuring concentration of hyaluronidase based on flow velocity of hydrogel composite membrane |
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| CN112964768A (en) * | 2021-03-23 | 2021-06-15 | 海南微氪生物科技股份有限公司 | Bst DNA polymerase electrochemiluminescence determination method |
| CN112980925A (en) * | 2021-04-06 | 2021-06-18 | 厦门大学附属厦门眼科中心有限公司 | MicroRNA detection method based on DNA hydrogel electrochemiluminescence system |
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